In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET). Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM). For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV)-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.Protein-protein interactions can be studied  Bacteria have long been regarded as vesicles filled with proteins without any internal organization. However, the cytosol of bacterial cells is densely crowded , so a hiin vitro methods (e.g. Surface Plasmon Resonance). These techniques are practical for screening potential interaction partners or to study binding affinities. In order to gain insight into the in vivo dynamics of these interactions, the in vivo method needs to produce time-resolved information. Förster Resonance Energy Transfer (FRET) allows time-resolved inspection of protein-protein interactions. Single cell FRET analysis allows for the investigation of individual differences of protein-protein interactions, rather than studying an average FRET efficiency of a population.Previously, techniques for studying protein-protein interactions were either indirect  or Fluorescence microscopy only allows identification of protein co-localization due to its limited resolution. FRET is the non-radiative energy transfer from an excited donor fluorophore to an acceptor fluorophore that can only occur when donor and acceptor are in very close vicinity of each other (<10 nm). FRET is therefore a useful tool for proving interactions via excitation of the donor fluorophore and measuring emission of the acceptor molecule –8.Escherichia coli [Bacillus subtilis competence machinery [FRET was first described by Förster –11 and fhia coli , and theachinery . For a sachinery . AnotherIn this study the FRET efficiency was determined in two ways: the first method is via the detection of sensitized emission : the meaet al. [Frequency Domain Fluorescence Lifetime Imaging Microscopy (FD-FLIM) ,17–19 alet al.  and the B. subtilis [amyE locus, expressed, and tested for FRET properties. The FPs were covalently linked with a linker containing a TEV protease recognition sequence. An inducible TEV protease gene was co-cloned with the FRET-pair allowing conditional high/low FRET efficiency situations. FRET efficiency was determined via sensitized emission and via a prototype MEM-FLIM system. The currently available FD-FLIM systems make use of an image intensifier, of which the photocathode is modulated at high frequencies (MHz). The image intensifier is typically the limiting factor for the spatial resolution, and is susceptible to damage by high light intensities. Therefore, FLIM recording is difficult to automate and is vulnerable. The prototype MEM-FLIM system used here modulates the CCD-sensor of the detector directly at the pixel level [Here, we investigated which FRET couple is best suited for dynamic protein-protein interaction studies in single cells of the Gram-positive model bacterium subtilis . Severalel level  resultinEscherichia coli MC1061 was used for cloning. All strains were cultivated on LB (Lysogeny Broth) medium at 37°C, supplemented with 100 μg/ml ampicillin or 100 μg/ml spectinomycin when appropriate. For the time-lapse experiment a chemically defined medium was used.The strains, plasmids, and oligonucleotides used in this study are listed in DNA purification, restriction and ligation were done as described before . Fast DiB. subtilis integration vector, pDOW01, was constructed by modifying pDR111 (kind gift of David Rudner). Both vectors integrate chromosomally in the amyE locus. The BglBrick assembly standard [In this study, a new standard  was intrEcoRI, BglII, BamHI and XhoI restriction sites, always remains present during cloning to realize up- or downstream insertion of a new part into an existing construct. The BglBrick cloning strategy is adapted from the BioBrick cloning method [Further, a TEV protease recognition site (ENLYFQG) coding sequence was inserted as a linker in the middle of the BglBrick cloning site. The BglBrick cloning site, with g method .E. coli was replaced with the ORI for E. coli from pUC18 to remove the BglII and XhoI restriction sites, the AmpR was removed, the BglBrick [To create pDOW01 from pDR111 the following changes were made: the ORI for BglBrick  restrictamyE gene and the spectinomycin marker from pDR111 (from 115–4707) and the E. coli origin of replication (1888–2583) from the pUC18 vector were PCR amplified including the NcoI and SpeI restriction sites in the primer sequences to combine the fragments into a pDR-pUC-hybrid , with the following changes. The XhoI, BglII and EcoRI sites were removed by point mutation. The SalI site was removed and in the upstream direction a RBS with a seven bp spacer to the start codon was inserted. The start codon was followed by the BglBrick prefix, the TEV-protease recognition site, the BglBrick suffix, two stop codons, and a strong terminator sequence. The IPTG-inducible Phyper-spank promoter from pDR111 remained unchanged. The synthetic DNA was inserted into the pDR-pUC-hybrid by replacing the original 422 bp between the SphI- and PstI-sites with the 529 bp synthetic DNA via restriction and ligation. This final cloning step resulted in our basic cloning vector, pDOW01 , Venus http://www.microscopyu.com/).pDOW01 was used as basic vector to construct the following FRET pairs and their donor and acceptor only counterparts: Cerulean-Venus, GFP-mCherry, GFP-mKate2, GFP-tagRFP, and Venus-mCherry. Cerulean and Venus are improved versions of CFP and YFP ,26. The EcoRI, BglII) in the forward primer and the BglBrick suffix  in the reverse primer for insertion into pDOW01  xylose was added to one part to induce the TEV-protease. Cells were grown for 2 more hours at 37°C and 2 ml of cell culture was centrifuged  and resuspended in 200 μl 50 mM Tris-Cl pH 7.4. Cell lysis was achieved by adding a small spatula tip of glass beads  to the mix, followed by two times one minute mini-bead beating . After centrifugation  the supernatant was carefully transferred to clean tubes and stored at -20°C. 30 μl supernatant supplemented with SDS-loading buffer was boiled at 80°C for 10 minutes and loaded on a 12% SDS-PAGE gel. After completion of electrophoresis the gel was transferred to a PVDF Western Blotting membrane (Roche. One hour at 80 mA), followed by blocking with 5% (w/v) skim milk (Oxoid) in PBST  Tween20, pH 7.3) overnight at 4°C. The PVDF-membrane was washed three times 15 minutes in PBST and incubated with PBST with 5% skim milk and a 1:10.000 dilution of anti-GFP  for two hours at room temperature. The membrane was washed three times 15 minutes in PBST and incubated with PBST supplemented with 1:5.000 goat-anti-rabbit Ig-Horseradish Peroxidase (Amersham Biosciences) for 1.5 hour at room temperature. Subsequently, the membrane was washed three times, gently dried with tissue papers and incubated for two minutes with 2 ml of ECL detection reagent . Signal visualization was done with a Molecular Imager ChemiDoc XRS+ (BioRad).An overnight culture of Microscope imaging for sensitized emission experiments was done using a Personal DeltaVision microscope system , with Softworx 3.6.0 software. The microscope was equipped with an Olympus IX71 inverted microscope body, a 100x phase contrast objective (Olympus PlanApo 1.40 NA), a CoolSNAP HQ2 camera (Princeton Instruments), a 300W Xenon light source, and filters for imaging of CFP , GFP , and mCherry  from Chroma. For Cerulean and Venus the CFP/YFP/mCherry polychroic mirror was used  and for GFP, mCherry, mKate2, and tagRFP the GFP/mCherry polychroic mirror was used . Images were captured using 0.2s light exposure with 32% light transmission for every combination of filters. The FRET channel was set in the software by using the CFP or GFP excitation filter and the GFP or mCherry emission filter.B. subtilis stocks and grown overnight at 37°C. The next morning the cultures were diluted 1 to 50 to an approximate OD600 of 0.03 in fresh LB medium and grown for two hours at 37°C at 225 rpm. The cells were induced with 0.1 mM IPTG and one part of the culture containing the two fluorophores was transferred into a new bottle containing 1% (w/v) xylose to induce the TEV-protease. After an additional two hours of incubation, cells were transferred to a microscope slide containing 1% (w/v) agarose to immobilize the cells and fluorescence intensity was measured with the wide-field microscope described above for FRET detection via sensitized emission. The same sample preparation method was applied for the FLIM experiments described below.LB medium was inoculated from -80°C 4)2SO4, 14 g K2HPO4, 6 g KH2PO4, 1 g Na3citrate.2H2O, 0.27 g MgSO4.7H2O, 20 mg casamino acids (Formedium), 20 mg L-tryptophan and 5 g D-fructose) and grown overnight at 37°C, 225 rpm. The next morning the culture was diluted to an OD600 of 0.08 in fresh CDM, grown for 5–7 hours at 37°C and 225 rpm to an OD600 of approximately 0.7. Now (0.35/OD)*250 = 125 μl of cells were diluted in 500 μl fresh medium and 2 μl was transferred to a slide with 1.5% low-melting point agarose (Sigma) in CDM and 0.1 mM IPTG. When the TEV-protease gene should be expressed 1% (w/v) xylose was added to the slide medium as well. To make sure that the fluorophores were present right from the start of the time-lapse experiment, IPTG  was added to the liquid culture one hour before transfer to the agarose slide. And in case the TEV-protease should be expressed also 1% (w/v) xylose was added to the liquid medium. The agarose slide was divided in three columns, separated by air cavities; one for B. subtilis DOW5 (GFP only), one for B. subtilis DOW13 (tagRFP only), and one for B. subtilis DOW23 (GFP-tagRFP).Time-lapse microscopy was done as described previously . BrieflyTime-lapse was done for 16 hours with a 15 minute interval. Of every strain the same four pictures were taken: phase contrast (GFP transmission light), FRET , donor (GFP excitation and emission), and acceptor (mCherry excitation and emission). In all cases the light exposure time was 0.2s and the light transmission was 32%. The microscope setup was the same as above except for the light source, which was now solid state TruLight Illumination . The microscope had a software-controlled stage to visit selected points routinely during a time-lapse experiment and the DeltaVision UltimateFocus was used to keep cells in focus.B. subtilis cells with only the donor, only the acceptor and with the donor and acceptor  was used to measure the pixel intensities of the B. subtilis cells and Microsoft Excel to process the data. First, the background pixel intensity was subtracted from the cellular pixel intensity and four correction factors were calculated to correct for the spectral overlap .The frequency domain MEM-FLIM system used (Lambert Instruments B.V.), consistet al. [Phase contrast images were taken using transmission light and 0.1s exposure time. FLIM data was collected using 0.7s exposure time and modulated LED light for excitation. Fluorescence lifetime data was collected using a modulation frequency of 40 MHz. For obtaining single cell fluorescence lifetime data the additional 1.5x magnification on the Nikon Microscope was used and the exposure time for collecting fluorescence images was extended to 1.5s. The LI-FLIM software was used for calculating the fluorescence lifetimes from phase shift data, see Zhao et al.  for detaDOW05), and from the strain with donor and acceptor (DOW23) with and without induction of the tev protease encoding gene.Acceptor photobleaching was performed as described previously . BrieflyThe FRET efficiency can be calculated with DA is the donor fluorescence intensity in presence of the acceptor and IDA* is the donor fluorescence intensity after photobleaching the acceptor.IB. subtilis using fluorescence microscopy. Therefore, the suitability of various fluorescent proteins (FPs) for FRET purposes in B. subtilis was tested by expressing them pairwise and covalently linked. The FPs tested here were Cerulean , Venus  cyan FP ), and sf cyan FP  as donor cyan FP ), and mKtor pDOW , which ator pDOW .tev gene, the heterodimer was efficiently cleaved into the monomers GFP and tagRFP (lane 4). Expression of the tev gene was a bit leaky, resulting in the presence of monomeric GFP without induction of tev expression (lane three). The band around 37 kDa in lane three is assumed to be a degradation product of the dimer. Overall, these results show that induction of the tev-protease gene results in an efficient separation of the FP pair  emission was lower when FRET occurs than when GFP and tagRFP are uncoupled by TEV-cleavage. The pre-bleach fluorescence intensities of GFP were: 362 AU without TEV protease vs. 437 AU when TEV was produced. Moreover, after acceptor photo bleaching, an increase in donor fluorescence was observed when the GFP-tagRFP was coupled: the fluorescence intensities of GFP were 410 AU (13% increase) when GFP-tagRFP was coupled vs. 429 AU (1.8% decrease) when GFP-tagRFP was uncoupled. Acceptor photobleaching increases the GFP fluorescence with 13% when the fluorophores were linked to each other. When the fluorophores were uncoupled by the TEV-protease, the donor fluorescence was approximately the same before and after photobleaching (437 vs. 429), which is a good control for this method.The FRET efficiency E see  is .The FRET efficiency can be calculated from the fluorescence lifetimes with Cells containing GFP-tagRFP were used to study the usability of the prototype MEM-FLIM system for FRET-FLIM measurements at the single bacterial cell level . In the in vivo examination of protein-protein interactions. It has been successfully applied for studying the sensor kinases CitA and DcuS in E. coli [E. coli [B. subtilis have been studied via acceptor photobleaching [E. coli has been studied extensively with acceptor photobleaching as well [Intermolecular FRET analysis allows  E. coli  and the [E. coli . The proleaching  and the  as well , but accin vivo FRET analysis in B. subtilis. FRET detected via sensitized emission showed that, out of the pairs tested, the GFP-tagRFP pair is the best candidate for FRET purposes in B. subtilis, based on the relative brightness and the quantum yield of tagRFP (see also B. subtilis is low for GFP-mCherry using both sensitized emission and FLIM (Tables Here we studied which FRET-pair would be a suitable candidate for see also  section.M Tables  and 7, dM Tables . DivisioM Tables . HoweverM Tables  and 7. IGFP-mCherry is often used as a FRET pair in interaction studies with high FRET efficiencies. In this study it might be that the properties of the linker prevent proper orientations of both fluorophores resulting in poor FRET efficiencies for this FRET-couple. FRET efficiency is dependent on three criteria for obtaining FRET. Of those criteria only the spectral overlap is independent of the construct used. The fluorophore distance and relative orientation of the donor and acceptor molecules depend on the linker sequence. Therefore, the FRET efficiencies reported here reflect the situation with the TEV-protease cleavable linker. FRET-efficiencies in earlier work range from 4 to 46% ,16,40,43Individually, the fluorescent proteins are efficiently produced  and, wheThe FRET-FLIM set-up used here allows measurements of FRET-efficiency on a single bacterial cell level . This sh


Aspergillus flavus infects crops and produces aflatoxin. Pichia anomala, which is a biocontrol yeast and produces the major volatile 2-phenylethanol (2-PE), is able to reduce growth of A. flavus and aflatoxin production when applied onto pistachio trees. High levels of 2-PE are lethal to A. flavus and other fungi. However, at low levels, the underlying mechanism of 2-PE to inhibit aflatoxin production remains unclear. In this study, we characterized the temporal transcriptome response of A. flavus to 2-PE at a subinhibitory level (1 µL/mL) using RNA-Seq technology and bioinformatics tools. The treatment during the entire 72 h experimental period resulted in 131 of the total A. flavus 13,485 genes to be significantly impacted, of which 82 genes exhibited decreased expression. They included those encoding conidiation proteins and involved in cyclopiazonic acid biosynthesis. All genes in the aflatoxin gene cluster were also significantly decreased during the first 48 h treatment. Gene Ontology (GO) analyses showed that biological processes with GO terms related to catabolism of propionate and branched-chain amino acids  were significantly enriched in the down-regulated gene group, while those associated with ribosome biogenesis, translation, and biosynthesis of α-amino acids were over-represented among the up-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that metabolic pathways negatively impacted among the down-regulated genes parallel to those active at 30 °C, a condition conducive to aflatoxin biosynthesis. In contrast, metabolic pathways positively related to the up-regulated gene group resembled those at 37 °C, which favors rapid fungal growth and is inhibitory to aflatoxin biosynthesis. The results showed that 2-PE at a low level stimulated active growth of A. flavus but concomitantly rendered decreased activities in branched-chain amino acid degradation. Since secondary metabolism occurs after active growth has ceased, this growth stimulation resulted in suppression of expression of aflatoxin biosynthesis genes. On the other hand, increased activities in degradation pathways for branched-chain amino acids probably are required for the activation of the aflatoxin pathway by providing building blocks and energy regeneration. Metabolic flux in primary metabolism apparently has an important role in the expression of genes of secondary metabolism.The saprophytic soil fungus  Aspergillus flavus, which is a common plant and an opportunistic human pathogen, can produce the carcinogenic aflatoxin. Contamination of crops such as corn, cotton, peanuts, and tree nuts by aflatoxin poses a great food safety risk especially in developing countries due to ineffective farming practices and the lack of proper storage facilities. Aflatoxin contamination can also result in devastating economic losses because of strict regulations on dissemination of contaminated products  AF36 [® . In fielPichia anomala WRL-076 is the only biocontrol yeast that has been shown to inhibit growth and aflatoxin production of A. flavus [Candida albicans [Kluyveromyces marxianus [Saccharomyces cerevisiae [Kloeckera apiculata [Penicillium italicum, which causes postharvest citrus decay [. flavus . 2-PE isalbicans , Kluyverarxianus , Saccharrevisiae  and Kloepiculata  also prous decay . The undus decay .A. flavus treated by a subinhibitory concentration (1.0 µL/mL) of 2-PE and to examine whether changes in the expression of specific genes of certain metabolic pathway had a bearing on inhibition of aflatoxin production. At this low concentration, 2-PE mostly stimulated fungal growth as evidenced by gene ontology (GO) enrichment analyses showing the increased structural constituent of ribosome and an active translation (α-amino acid biosynthesis). The outcomes, along with a decrease in the degradation of branched-chain amino acids, were correlated with the suppression of all aflatoxin pathway gene expression.A better understanding of the mode of action of 2-PE at low concentrations, a scenario likely to be encountered in field applications of the biocontrol yeast, is critical to the development of an effective biocontrol formulation. At subinhibitory levels to fungal mycelial growth , how 2-PA. flavus NRRL3357. Among these reads about 96.9% were located in the exon regions and 3.1% were located in the intron regions. Volcano plots derived from the 24 h, 48 h and 72 h gene expression data showing original p-values on the y-axis and fold change on the x-axis was generated  correction of h period . ClusterA. flavus, all genes in the aflatoxin biosynthesis gene cluster were examined -rRNA from 5.8S rRNA and LSU (large subunit)-rRNA from tricistronic rRNA transcript , endonucleolytic cleavage to generate mature 3′-end of SSU-rRNA from , translation, and biosynthesis (anabolism) of α-amino acids. p-values.The specific GO terms enriched in the down-regulated gene group were mainly in the biological process category. These included catabolism of branched-chain amino acids of valine, leucine and isoleucine, and in the biosynthetic pathway of polyketide-derived sterigmatocystin and aflatoxin a. The spThe KEGG pathway analysis confirmed that the amino acid metabolic pathways identified in the down-regulated gene group were consistent with the enriched GO terms in the biological processes category including those involved in metabolism branched-chain amino acids . In addiA. flavus. Previous results indicate that 2-PE (1.0 µL/mL) used in the present study has no discernible adverse effects on fungal growth and does not disrupt membrane integrity [aflR and aflJ, which are required for transcriptional activation of other aflatoxin biosynthetic genes [aflR and aflJ expression at 48 h and 72 h also correlates with a lesser extent of decrease in other gene expression. Global regulatory genes such as veA and laeA control the expression of aflR and aflJ [veA and laeA genes, the decrease is not significant (A. flavus likely results from a broad change in physiology and metabolism (see below).2-PE at high concentrations (0.3%–0.5%) is lethal to bacteria and fungi ,16,17,18ic genes ,20. The and aflJ ,22. Althnificant . The decC- and N-containing macromolecules. In the up-regulated gene group GO terms associated with ribosome synthesis, translation and anabolism of α-amino acids are enriched. Taken together, the 2-PE treatment promotes active fungal growth. The KEGG metabolic pathway analyses of the GO-enriched down- and up-regulated gene groups also support this explanation , and catabolism of carboxylic acid and branched-chain amino acids  are over-represented. These results are indicative of a metabolic flux being channeled toward primary metabolism for synthesis of lanation . Althouglanation . The degat 37 °C . The rol. flavus . The lat. flavus . Asparag alanine . As a suoduction . In thiset al. (2014) have reported that a short 2 h exposure of Penicillium italicum to a lethal concentration of 2-PE (1.5 μL/mL) induces subcellular changes in hyphae characteristics by vacuolation, degradation of organelles, and leakage in the outer membrane of mitochondria. In the corresponding transcriptomic analysis, the expression of genes associated with ribosome and amino acid biosynthesis is down-regulated and they conclude that inhibition of the amino acids and protein biosynthesis is the cause of the lethal effect of 2-PE [A. flavus totally opposite changes; it promotes protein synthesis for growth as evidenced by the GO enrichment analysis and the result that expression of about 70% (46/67) of genes encoding known or putative ribosomal proteins is increased significantly in the first 48 h , but it inhibits secondary metabolism, like the decreased expression of all genes in the aflatoxin and cyplopiazonic acid gene clusters. Studies have shown that plant volatiles of aldehydes, ketones, and alcohols from cotton leaf [A. flavus or closely related A. parasiticus is decreased or abolished when exposed to sublethal levels of volatile(s) remains unclear. Nonetheless, this study shows that active growth and decreased activities in the metabolism of specific amino acids have a bearing on the decrease in all aflatoxin gene expression in the 2-PE treated A. flavus.The mode of action of 2-PE includes inhibition of synthesis of DNA ,15,30 an of 2-PE . In thiston leaf , corn [3ton leaf , and soyton leaf  either aA. flavus treated by decanal [con (conidiation) genes were found not to be conidia-specific but are associated with stresses because they are highly expressed in sclerotia, which are hardened hyphal aggregates for survival at unfavorable environments. In addition, Con proteins contain a characteristic stress-response KGG repeat motif. Consistently, con transcripts are not detected in growing mycelia in Neurospora crassa [con genes in the 2-PE treated A. flavus . A fresh spore suspension was prepared in a 0.05% Tween 80 solution. The NYDB growth medium consisted of nutrient broth 8 g, yeast extract 5 g, and glucose 10 g/L. Aliquots of the spore suspension were inoculated into 20 mL of NYDB in a 125 mL flask to a final concentration of 105/mL. For the treatment set, 2-phenylethanol  was added to a final concentration of 1 µL/mL. For the control and treatment sets three cultures of each were grown at 28 °C on a rotary shaker at 150 rpm. At 24 h, 48 h and 72 h after inoculation, mycelia were harvested, rinsed with cold DEPC-treated water (0.1% solution), dried by vacuum suction, and ground in a chilled mortar with liquid nitrogen until a fine powder was achieved.Total RNA isolation was carried out using RNeasy Plant Mini Kit . The RNA samples were treated with Ambion TURBO DNA-free DNase . The purity and concentrations of RNA were examined by a ND-1000 Spectrophotometer . RNA quality was assessed with an Agilent 2100 Bioanalyzer  and all were with RIN (RNA Integrity Number) between 6.9 and 7.8. Samples were stored in a −80 °C freezer until use. Total RNA was processed using an Illumina TruSeq RNA Sample Prep kit, following the manufacturer’s instruction . After various quality control procedures, individual RNA-Seq libraries were pooled at an equal molar ratio based on their respective sample-specific 6-bp adaptors. Pooled RNA-Seq libraries were then sequenced at 50bp/sequence read using an Illumina HiSeq 2000 sequencer as described previously . Raw sinA. flavus NRRL3357 [Mapping at 80% identity fraction and 80% length fraction using the RNA-Seq module of CLC Genomic Workbench version 8 was performed . All reaNRRL3357  and exprNRRL3357  were cali.e., sample replicates, against those in another set of count samples. In comparison, Fisher’s Exact Test compares the counts in one sample against those of another. Total count filter cutoff number was set 5. FDR  corrected-p values were calculated from the original p-values [p-values was set to be false-positive (p < 0.05).The statistical analysis was performed using the Empirical Analysis of DGE  function of CLC Genomics Workbench, which implements the “Exact Test” for two-group comparisons . This mep-values . FDR is Blast2GO  was usedA. flavus. Metabolism of different amino acids in primary metabolism and secondary metabolism are associated with A. flavus growth, development, and aflatoxin production. Noticeably, aflatoxin production requires a higher activity in the catabolism of branched-chain amino acids. Likely, the end products of this degradation pathway such as acetate and propanoate not only serve as precursors that are channeled into aflatoxin biosynthesis but are also used for energy regeneration. Metabolic flux from primary metabolism can impact the expression of genes of secondary metabolism.Our functional genomics study shows that the inhibition of aflatoxin production by the low level of 2-PE results from its effect on promoting active growth of


Furthermore, simulations indicate that the drain current and field-effect mobility extraction are limited by the contact resistance. When the effects of contact resistance are de-embedded, a field-effect mobility of 129 cm2/V.s is obtained. This excellent result demonstrates the promise of top-down ZnO nanowire technology for a wide variety of applications such as high-performance thin-film electronics, flexible electronics, and biosensing.This paper studies the effect of atomic layer deposition (ALD) temperature on the performance of top-down ZnO nanowire transistors. Electrical characteristics are presented for 10-μm ZnO nanowire field-effect transistors (FETs) and for deposition temperatures in the range 120°C to 210°C. Well-behaved transistor output characteristics are obtained for all deposition temperatures. It is shown that the maximum field-effect mobility occurs for an ALD temperature of 190°C. This maximum field-effect mobility corresponds with a maximum Hall effect bulk mobility and with a ZnO film that is stoichiometric. The optimized transistors have a field-effect mobility of 10 cm The ID-VG characteristics were used as shown in Figure ∂ID/∂VG of the simulated characteristic, a maximum transconductance (gm) of 8.5 nS is obtained and a field-effect mobility of 8 cm2/V.s. These values are in good agreement with the measured transconductance gm of 10.4 nS and the measured field-effect mobility of 10 cm2/V.s. The effect of contact resistance on field-effect mobility can be de-embedded using the simulated characteristic for a device with a 10-Ω contact resistance in Figure 2/V.s is obtained. This value is significantly higher than the measured value of 10 cm2/V.s and demonstrates the potential for further improvements in the on-current through modifications to the contact resistance technology.To determine the effect of contact resistance only on the extraction of field-effect mobility, the simulated and measured linear 2/V.s) have been reported in passivated ZnO nanowire transistors [2/V.s) in unpassivated devices. The results presented in this work were obtained on unpassivated ZnO nanowire transistors. There may also be a scope to further increase the mobility in our devices by using surface passivation.Considerable research has been published in the literature on bottom-up ZnO NW FETs -33, withnsistors ,27 but m2/V.s and also corresponds with the maximum Hall effect mobility of 120 cm2/V.s. This result is explained by the good stoichiometry of the ZnO films at a deposition temperature of 190°C. The optimized field-effect mobility of 10 cm2/V.s is approximately ten times higher than can typically be achieved with thin-film amorphous silicon transistors. Furthermore, device simulations show that the field-effect mobility is limited by contact resistance and when this is de-embedded, the field-effect mobility increases to 129 cm2/V.s. It is clear therefore that top-down fabricated ZnO nanowire transistors show considerable potential for high-performance, transparent, thin-film electronics on either glass or polymer substrates.This paper has studied the effect of the atomic layer deposition temperature on the performance of top-down, ZnO nanowire field-effect transistors. The ZnO deposition temperature has been systematically varied, with all other deposition conditions kept constant. A deposition temperature of 190°C gives the maximum field-effect mobility of 10 cmThe authors declare that they have no competing interests.SMS carried out the study, experiments, and characterizations. NJD performed the simulation study. RG advised on the ALD deposition technique. PA, HMHC, and SMS drafted the manuscript. All authors read and approved the final manuscript.


The present research manipulated the fluency of unstudied items using masked repetition priming procedures during an explicit recognition test. Based on fluency-attribution accounts, which posit that familiarity can be driven by multiple forms of fluency, the relationship between masked priming-induced fluency and familiarity was investigated. We classified pictographic characters into High-Meaningfulness (High-M) and Low-Meaningfulness (Low-M) categories on the basis of subjective meaningfulness ratings and identified the distinct electrophysiological correlates of perceptual and conceptual fluency. The two types of fluency differed in associated ERP effects: 150–250 ms effects for perceptual fluency and FN400 effects for conceptual fluency. The ERPs of Low-M MP-same (items that were preceded by matching masked items) false alarms were more positive than correct rejections during 150–250 ms, whereas the ERPs of High-M MP-same false alarms were more positive than correct rejections during 300–500 ms. The topographic patterns of FN400 effects between High-M MP-same false alarms and Low-M MP-same false alarms were not different from those of High-M hits and Low-M hits. These results indicate that both forms of fluency can contribute to familiarity, and the neural correlates of conceptual fluency are not different from those of conceptual priming induced by prior study-phase exposure. We conclude that multiple neural signals potentially contribute to recognition memory, such as numerous forms of fluency differing in terms of their time courses. Recognition decisions can be subdivided into two expressions: familiarity and recollection. Familiarity refers to the impression that a particular item was encountered previously without the recall of relevant details. For example, a man looks familiar to us, but we cannot recollect where we met him. Recollection, in contrast, implies that the spatiotemporal context or other details about the prior event can be recalled, such as the man’s name or the color of the jacket he was dressed in when we encountered him . We therefore predicted that ERPs related to perceptual fluency would occur earlier than FN400 effects associated with conceptual fluency. We also predicted that when familiarity was driven by different forms of fluency, distinct ERPs will be exhibited.The present research seeks further evidence to clarify these issues by using a unique type of stimuli: ancient Chinese characters, including oracle bone scripts and bronze inscriptions. These stimuli were first used in a memory study conducted in our laboratory  participated in the experiment. All were right-handed and reported normal or were corrected to normal vision. Data from one subject was collected but excluded due to excessive ocular artifacts and/or electrode drift . All subjects signed an informed consent and were paid for their participation. None of the subjects majored in History or Ancient Chinese Language and none had any background in learning Chinese oracle and bronze inscriptions. This research was approved by the Human Research Ethics Committee at Capital Normal University.The stimuli consisted of 600 ancient Chinese characters. An additional 25 items were used as fillers. These hieroglyphs cannot be understood by contemporary people without professional knowledge of history and the ancient Chinese language . In each test phase, participants completed a recognition test in which 40 studied items were presented again, along with 80 novel items in pseudo-randomized order (no more than three items of the same old/new type occurred consecutively). Participants were misinformed that the ratio of old to new items was 1:1. During each test phase, 50 new trials were MP-same , and 30 new trials were MP-different . All old trials were MP-different.Masks took the form of random lines the same size as the test stimuli. One forward mask and one backward mask sandwiched each prime item in the test phase. Participants were not informed about the presence of the masked items. They were only told that these “flickers” would be used to obtain a baseline measure of brain activity. It was emphasized that in order to have the best memory performance, they needed to focus on the test items. Before the formal experiment, there was a practice section for participants to familiarize themselves with the procedure.Each study trial began with a fixation cross, followed by a 2000 ms study item and then a fixation cross. The ISI was randomized between 1000 ms and 2000 ms. The task was the same as a previous study conducted in our lab, participants were instructed to rate the meaningfulness level of each item using a 4-point scale, with: 1 = highly meaningful, 2 = relatively meaningful, 3 = relatively meaningless, 4 = almost meaningless. Instructions were to press 1 if an item looked like nameable objects, animals or scenes, 2 if it evoked an intangible meaning or connotation, 3 if it only evoked minimal meaning with effort, or 4 if the item could not evoke any meaning. For example, the third hieroglyph in the top row in Figure The test phase followed the study phase in each block after a break of 30 s, during which participants counted backward by threes. Each test phase was preceded by two practice trials . Each test trial began with a fixation cross, the duration of which was randomized between 700 ms and 1200 ms; then a 40 ms forward mask was presented, followed by a 40 ms matching or non-matching prime item, and a 40 ms backward mask. A fixation cross was then shown again, the duration of which was randomized between 400–500 ms, followed by an 800 ms test item and then a fixation cross.Participants indicated how confident they were in judging an item as studied (or unstudied) by pressing a single button: button 1 = high confidence old (if they were sure that the item was studied); button 2 = low confidence old (if they thought that the item was studied but without confidence); button 3 = low confidence new (if they thought that the item was unstudied but without confidence); button 4 = high confidence new (if they were sure that the item was unstudied). Both speed and accuracy of response were emphasized. Buttons 1–4 corresponded to the index and middle fingers of the right and left hands. The response buttons were counterbalanced between participants.After the 5 study-test blocks, there was a re-rating phase where participants were asked to rate the meaningfulness of all the items according to the 4-point scale. This allowed meaningfulness ratings to be determined for new items in the experiment. EEGs were not recorded in this phase. Items given meaningfulness ratings of 1 or 2 were categorized as High-M; Items given meaningfulness ratings of 3 or 4 were categorized as Low-M. Thus, the meaningfulness level of old items was rated twice (the study phase and the re-rating phase) while that of the new items was rated once (the re-rating phase). For the sake of consistency, the trials from the test phase were subdivided into High-M and Low-M categories based on the results from the re-rating phase. Given that the meaningfulness ratings for each item were highly variable across subjects, these two categories were created separately for each subject.After the re-rating phase, participants were asked whether they had noticed something presented during the “flickers”. If the answer was “yes”, they were further asked what they had perceived. In the debriefing, 6 of the 16 participants reported that they had noticed something presented during the “flickers”, and the number of “noticed” trials was less than 10 in a test block. They reported that the “flickers” were ancient Chinese characters similar to the test items. Five of the 6 participants reported that they had not noticed whether the “flickers” and the later test items were the same; 1 of them reported that occasionally the “flickers” ancient Chinese characters were the same as the later test items, and the number of the “same” trials was less than 5 in a test block. Participants were also asked what they imagine the characters as when they rated the meaningfulness levels of the items.mean = 3.9%, SE = 0.01). EOG blink artifacts were corrected using a linear regression estimate. Repeated-measures ANOVA included Greenhouse-Geisser corrections when necessary. The alpha level was 0.05.Electroencephalographic recordings were made from 62 scalp sites using Ag/AgCl electrodes embedded in an elastic cap. Electrode locations adhered to the extended international 10–20 system. These electrodes were referenced to the left mastoid during recording and re-referenced to the average of the right and left mastoid offline. Impedance was less than 5 kΩ. EEG signals were filtered with a band-pass of 0.05–40 Hz and sampled at a rate of 500 Hz. Each 1000 ms averaging epoch began 100 ms prior to stimulus onset. Baseline corrections were performed using mean amplitudes of pre-stimulus onset. Trials containing baseline drift exceeding ±75 μV were rejected . The analysis of behavioral results was focused on response time and accuracy. The analysis of ERP results included three steps. First, we investigated ERPs related to fluency from study-phase by comparing the High-M hits with Low-M hits. The mean numbers of artifact-free trials for High-M hits and Low-M hits were 76 and 51. Second, in order to examine the masked priming effects, we collapsed across response types to examine overall differences between MP-same and MP-different unstudied trials for High-M and Low-M items. The mean numbers of artifact-free trials for the four conditions were as follows: High-M MP-same 108, High-M MP-different 78, Low-M MP-same 129, Low-M MP-different 75. Third, we used the unstudied item trials to isolate the neural correlates of familiarity induced by masked repetition priming. The relationship between familiarity and fluency was investigated by comparing false alarms  with correct rejections. One participants were removed due to fewer than 16 artifact-free trials for the Low-M MP-different false alarms condition. For the remaining 15 subjects, the mean numbers of artifact-free trials for false alarms and correct rejections were as follows: High-M MP-same 48, 63; High-M MP-different 28, 42; Low-M MP-same 43, 87; Low-M MP-different 25, 51.Based on the previous research on recognition memory and fluency  = 8.813, p = 0.01). The main effect of masked priming was significant  = 21.426, p < 0.001), as well as the interaction of meaningfulness and masked priming  = 7.97, p = 0.013). The simple effect analysis revealed that the masked priming was significant in High-M condition  = 37.301, p < 0.001) and Low-M condition  = 4.91, p = 0.043), although the magnitude of masked priming  for High-M were greater than that for Low-M (t(15) = 2.823, p = 0.013). Average response times for MP-same and MP-different stimuli as a function of meaningfulness is shown in Figure Response time in the recognition test for each condition is shown in Table F = 20.654, p < 0.001), reflecting a greater proportion of false alarms for High-M relative to Low-M. The main effect of confidence was also significant  = 11.143, p = 0.004). The main effect of masked priming was not significant  = 0.511, p = 0.485). In addition, a marginal significant interaction between meaningfulness and confidence emerged  = 3.391, p = 0.085). Further analyses indicated that there was a stronger effect of meaningfulness on high-confidence relative to low-confidence false alarms.Mean percentage of responses in each condition is shown in Table A priori time window of 300–500 ms was selected according to previous literatures  with Low-M hits . F = 13.218, p = 0.001). The simple effect analysis indicated that the two conditions were significantly different only at frontal (t(15) = 2.953, p = 0.01).A 2 × 3 ANOVA was conducted with meaningfulness (High-M hits/Low-M hits) and cluster  factors in the 300–500 ms interval, The interaction effect of meaningfulness and cluster was significant  and cluster  at intervals of 150–250 ms and 300–500 ms.F = 1.724, p = 0.209). The masked priming × cluster effect was significant  = 6.227, p = 0.016). The simple effect analysis indicated that the MP-same Low-M trials were more positive than MP-different Low-M trials only in the frontal region (t(15) = 2.463, p = 0.026).For the 150–250 ms interval, the main effect of masked priming was not significant  = 2.907, p = 0.109), and neither was the masked priming × cluster interaction  = 0.63, p = 0.466).For the 300–500 ms interval, the main effect of masked priming was not significant  and cluster  at intervals of 150–250 ms and 300–500 ms.F = 0.23, p = 0.638), the interaction of masked priming and cluster was not significant  = 0.588, p = 0.487).For the 150–250 ms interval, the main effect of masked priming was not significant  = 6.653, p = 0.021), the interaction of meaningfulness and cluster was not significant  = 0.468, p = 0.566).For the 300–500 ms interval, the main effect of masked priming was significant . As shown in Figure Formal ERP comparisons across false alarms and correct rejections over the 150–250 and 300–500 ms latency intervals were performed for these four conditions. The 2 × 3 ANOVA were conducted with the factors of response types (FAs/CRs) and cluster .For the 150–250 ms interval, the main effect of response types (FAs/CRs) was only significant for Low-M MP-same condition. For the 300–500 ms interval, the main effect of response types (FAs/CRs) was only significant for High-M MP-same condition. The detailed results were summarized in Table In order to examine the differences between perceptual fluency and conceptual fluency as they contributed to familiarity, the ERPs for High-M MP-same false alarms and Low-M MP-same false alarms were compared at 150–250 ms and 300–500 ms intervals Figure . The HigF = 0.365, p = 0.555), and neither was the interaction between meaningfulness × cluster  = 0.075, p = 0.822).For the 150–250 ms interval, a 2 × 3 ANOVA with the factors of meaningfulness (High-M MP-same/Low-M MP-same) and cluster  was conducted. The main effect of meaningfulness was not significant  = 4.572, p = 0.051). The meaningfulness × cluster interaction was marginal significant  = 3.272, p = 0.083). The simple effect analysis indicated that the effect of meaningfulness was more robust at frontal.For the 300–500 ms interval, a similar 2 × 3 ANOVA was conducted. The main effect of meaningfulness was marginal significant  = 0.623, p = 0.679). This result indicated that the scalp distribution of the FN400 effects did not differ between the two conditions.The 300–500 ms positive differences for High-M hits compared to Low-M hits Figure  might alThe present research manipulated the fluency of unstudied items using masked repetition priming procedures during an explicit recognition test. Based on fluency-attribution accounts, which posit that familiarity can be driven by multiple forms of fluency, the relationship between masked priming-induced fluency and familiarity was investigated. We classified pictographic characters into High-M and Low-M categories on the basis of subjective meaningfulness ratings and identified distinct electrophysiological correlates of perceptual and conceptual fluency. The two types of fluency differed in associated ERP effects- 150–250 ms effects for perceptual fluency and FN400 effects for conceptual fluency. The ERPs of Low-M MP-same false alarms were more positive than those of correct rejections during 150–250 ms, while the ERPs of High-M MP-same false alarms were more positive than those of correct rejections during 300–500 ms. The topographic patterns of FN400 effects between High-M MP-same false alarms and Low-M MP-same false alarms were not different from that of High-M hits and Low-M hits. These results indicated that both forms of fluency could contribute to familiarity, and the neural correlates of conceptual fluency were not different from those of conceptual priming induced by prior study-phase exposure.Response time results indicated that masked priming occurred for the unstudied items; the response time of MP-same items was faster than that of MP-different items, especially for High-M items. The response time of High-M items was faster than that of Low-M items. These suggested that the mechanism behind the masked priming effect was processing fluency.A difference between our results and those of Lucas et al.  is the aSome studies suggested that familiarity was indexed by FN400 effect and recollection was indexed by LPC potentials , in which both perceptual and conceptual fluency contributed to familiarity. This ERP effect might signal a contribution of masked priming-induced conceptual fluency to familiarity. For MP-different items, perceptual and conceptual fluency did not change. There were no ERP differences between the false alarms and correct rejections at these two intervals. These results suggested that the contribution of perceptual and conceptual fluency to familiarity disassociated in different time courses; namely, familiarity induced by perceptual fluency appeared at 150–250 ms interval, and familiarity induced by conceptual fluency was indexed by the 300–500 ms effect—consistent with the latency of FN400.vs. Low-M hits) were obtained, whereas the ERP differences in the 150–250 ms interval were not significant. The scalp distribution of FN400 effects induced by masked priming  and those induced by study-phase exposure (High-M hits vs. Low-M hits) did not differ from each other. Thus, we found no evidence that the conceptual fluency induced by study phase exposure and that induced by masked-prime have different neural bases.We compared MP-same false alarms for High-M  with those for Low-M  categories in order to examine ERP effects associated with the contribution of conceptual fluency to familiarity. Similar FN400 potentials to FN400 effects reported above (e.g., High-M hits We did not employ the usual Remember/Know Judgments to dissociate recollection from familiarity and all our analyses were collapsed across confidence levels, leaving open the possibility that participants might have experienced a certain amount of false recollection because of the masked priming manipulation. Although most previous researches suggested that masked priming manipulation primarily affected familiarity but not recollection (e.g., Rajaram, Finally, by separating materials according to meaningfulness ratings, the present study emphasizes that the experience of familiarity could be driven by both perceptual and conceptual fluency and that the electrophysiological correlates of the contribution of perceptual and conceptual fluency to familiarity are different. Perceptual priming and conceptual priming respectively driven by perceptual and conceptual fluency are processed during different periods. This finding corresponds to theories of multiple memory systems Tulving, . HoweverThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


L-tyrosine methyl ester (L-TM) to the methyl ester of para- hydroxycinnamic acid (p-HCAM) using Rhodotorula glutinis yeast phenylalanine/tyrosine ammonia lyase  enzyme was successfully demonstrated for the first time; progress of the reaction was followed by spectrophotometric determination at 315 nm. The following conditions were optimized for maximal formation of p-HCAM: pH (8.5), temperature (37°C), speed of agitation (50 rpm), enzyme concentration (0.080 μM), and substrate concentration (0.50 mM). Under these conditions, the yield of the reaction was ∼15% in 1 h incubation period and ∼63% after an overnight (∼18 h) incubation period. The product (p-HCAM) of the reaction of PTAL with L-TM was confirmed using Nuclear Magnetic Resonance spectroscopy (NMR). Fourier Transform Infra-Red spectroscopy (FTIR) was carried out to rule out potential hydrolysis of p-HCAM during overnight incubation. Potential antibacterial activity of p-HCAM was tested against several strains of Gram-positive and Gram-negative bacteria. This study describes a synthetically useful transformation, and could have future clinical and industrial applications.Biotransformation of  PAL ; and the enzymes that show dual specificity toward L-Phe and L-Tyr are called PTAL , we also intend testing its potential application as a food additive .We have successfully demonstrated the direct one-step PTAL catalyzed synthesis of n plants , health n plants , and foon plants . TherefoRhodotorula glutinis yeast was used as the source of PTAL (E.C.4.3.1.26) enzyme. The isolated enzyme was obtained from Sigma–Aldrich Company .L-Phe, L-PM, L-Tyr, and L-TM were obtained from Sigma–Aldrich Canada Ltd . Authentic p-HCA and p-HCAM were procured from Fisher Scientific  and Frinton Laboratories , respectively. Deuterated solvents used for NMR studies were purchased from Acros Organics . All other commonly used chemicals, reagents, and solvents were of highest analytical grade; and were bought from Fisher Scientific .Authentic L-TM as the substrate was performed and formation of the product, p-HCAM, was followed spectrophotometrically, using a Cary 100 UV-vis spectrometer, by monitoring the wavelength at 315 nm. Initially, the PTAL reaction was carried out as follows: the reaction mixture (1.0 mL) containing 50.0 mM Tris-HCl buffer (pH 8.8), 1.0 mM L-TM, and 0.1 μM PTAL enzyme protein was incubated at room temperature for 60 min. At the end of the incubation period, the reaction was terminated by deactivating the enzyme with 20 μL of 12 M HCl. The reaction mixtures, in which substrate and enzyme were added, after termination of reaction, served as substrate and enzyme controls, respectively. PTAL activity is reported in terms of p-HCAM formation (measured as O.D. units at 315 nm). Different conditions for maximal p-HCAM production including pH, temperature, speed of mixing the reaction mixture, and the enzyme and substrate concentrations were then standardized when the reaction was carried out for 60 min.The PTAL assay using L-TM to p-HCAM was scaled up to a total volume of 5.0 mL , and the reaction was carried out for ∼18 h to ensure production of sufficient amount of the product for detection by NMR and FTIR. At the end of the incubation period, solvent buffer from the reaction mixture was removed by rotary evaporation.The PTAL catalyzed biotransformation of p-HCAM was soluble, whereas L-TM and PTAL were insoluble in CDCl3), and the solution was filtered to separate unreacted substrate and enzyme from the product. The filtrate containing p-HCAM was then characterized using solid state CP/MAS NMR carried out on a Bruker Avance NMR spectrometer with a 9.4 T magnet (400.24 MHz proton Larmor frequency and 100.65 MHz 13C Larmor frequency). Authentic p-HCAM in CDCl3 was characterized in a similar manner by NMR.The residue obtained at the end of the rotary evaporation step was treated with deuterated chloroform . KBr pellets were prepared using these specimens; and infrared absorption spectra of each were measured by absorption techniques using a Nicolet Impact 410 FTIR over a range of 4000–490 cm-1.Fourier Transform Infra-Red spectroscopy analysis was carried out to rule out potential hydrolysis of L-TM to p-HCAM using PTAL was carried out according to the protocol developed in our lab; and the residue obtained was dissolved in 50% ethanol. Unreacted L-TM and PTAL which were insoluble in ethanol were separated out by filtration, and the antibacterial activity of the filtrate containing p-HCAM was tested by cylinder-plate (agar-cup plate) diffusion method (Bacillus cereus and Staphylococcus aureus) and Gram-negative organisms . Each of the bacterial cultures was grown for 24 h at 37°C on 2.8% nutrient agar plates. Suspensions of each culture were prepared in sterile saline and then swabbed onto 3.8% Muller-Hinton agar plates. Various concentrations of p-HCAM solution  were tested for antibacterial activity against each culture. The Muller-Hinton agar plates were incubated for 24 h at 37°C and the zones of inhibition were measured.Biotransformation of n method  against The values reported are a mean of at least five independent determinations.p-HCAM and shown its potential antibacterial activity.In this study, we have developed a simple and rapid procedure for the production of L-TM and p-HCAM was tested in several buffer systems, reagents, and solvents (data not shown). Due to limited solubility of L-TM at low pH, PTAL activity was determined between 8.1 and 9.0 using 1 mM substrate concentration. Since L-TM and p-HCAM have distinct λmax at 275 and 315 nm, respectively; it can be concluded that interference of substrate with product determination is unlikely to happen. Further confirmation that substrate and enzyme do not contribute to absorbance values when determining the product was carried out by using appropriate substrate and enzyme controls during PTAL assay. Using an enzyme control was particularly important as the PAL/PTAL enzyme shows absorbance at 280 nm.The solubility of Several experimental conditions including pH, temperature, speed of mixing the reaction milieu, and enzyme and substrate concentrations were optimized when this reaction was carried out for an incubation period of 60 min.L-TM was totally insoluble in 0.1 M phosphate buffers at pH 7.0 and 8.0 but soluble in 0.1 M Tris-HCl buffer at pH 9.0, we carried out pH optimization studies using Tris-HCl buffer with increments of 0.1 units in the range 8.1–9.0. PTAL catalyzed transformation of L-TM to p-HCAM was maximal at pH 8.5 . We did not test pH exceeding 9.0; PAL has been reported to show a progressive gradual decline in activity at pH greater than 9.0 , it is evident that in the present study, optimal PTAL activity was obtained at 37°C and that there was progressive decline in enzyme activity at higher temperatures.From the curve of temperature versus enzyme activity (Table 1). These results correspond well with almost all studies on effect of agitation on PAL protein using L-Phe, L-PM, t-CA, and t-CM substrates; optimal enzymatic conversions have been obtained at ∼50–100 rpm (There was no pronounced effect on PTAL enzyme activity when the speed of agitating the reaction mixture during the incubation period was increased above 50 rpm (–100 rpm .A315nm/1.0 μM TAL) was maximal when 0.080 μM PTAL enzyme protein was used for assay . There was no substantial increase in PTAL enzyme activity beyond this concentration of enzyme protein. The enzyme activity for individual concentrations of L-TM is shown in Figure 1E. Unit Enzyme activity (A315nm/1.0 mM L-TM) was maximal when 0.50 mM L-TM was used for assay; PTAL enzyme activity decreased progressively below and above this substrate concentration.Unit Enzyme activity  was obtained from A315nm values by using a calibrated curve constructed with authentic p-HCAM drawn from a 0.010 M stock solution . The yield of p-HCAM increased to ∼63%, when the reaction was carried out overnight (∼18 h).The conditions standardized for the optimal production of L-TM to p-HCAM reported in this study is a novel reaction. NMR acquired data provides convincing evidence for the formation of the desired product, p-HCAM. The 1H  and 13C  NMR spectra of the product of our reaction and of authentic p-HCAM were identical. The analysis of NMR spectral peaks representing different protons and carbons of the product is shown in Scheme 1.The conversion of p-HCAM , it is further confirmed that the PTAL catalyzed biotransformation of L-TM to p-HCAM was successful. The FTIR spectral profiles reveal that product obtained in our study and authentic p-HCAM have nearly identical absorbance in the range 3700–2000cm-1; while authentic p-HCA gives a significantly different absorbance profile in this region. Therefore, we can conclude that there is negligible or no hydrolysis of p-HCAM to p-HCA during the incubation period of ∼18 h.From the striking similarities between FTIR spectra of the product of our reaction and authentic P. vulgaris and B. cereus (Table 3) when 5.0% p-HCAM (product of PTAL obtained in our lab) was loaded in the well of Muller-Hinton agar plate, were comparable with that obtained with standard antibiotic, Ampicillin (∼9 mm zone of inhibition). While substantial antibacterial activity was obtained against S. aureus, E. coli, and K. pneumoniae, the antibacterial effect against P. aeruginosa was negligible (we have not tried a concentration exceeding 5.0% p-HCAM in this study). Ethanol in which the product of our reaction was dissolved, did not contribute to the antibacterial activity (no zone of inhibition was obtained). The data obtained with 1.0, 3.0, and 5.0% concentrations of authentic p-HCAM  revealed similar results with each of the six strains used in this study; however, smaller zones of inhibition (data not shown in Table 3) were obtained, possibly due to the presence of some impurities.The zones of inhibition obtained with L-Phe, L-PM, and p-HCA ; and p-HCA has several applications in pharmaceutical, health, and food industries  was no exception. The only discrepancy was that we did not get a typical bell shaped curve for a plot of pH vs enzyme activity as reported in earlier studies  are similar to most results obtained with temperature studies on PAL/PTAL (including R. glutinis PAL/PTAL) that have been reported earlier, in that, the enzyme has been known to have maximal activity at 30–37°C, and that activity rapidly falls at temperatures equal to or greater than 40°C  could be substrate inhibition of the enzyme. Several enzymes have been reported to be inhibited by their substrates when the latter are in high concentration; such type of inhibition is regarded as a form of uncompetitive allosteric inhibition of the enzyme  obtained for S. aureus, E. coli, and P. aeruginosa are in good agreement with those obtained by p-HCAM was dissolved, contributed to the antibacterial activity. Since, p-HCAM is literally insoluble in aqueous media but freely soluble in organic solvents, and it is well known that a number of organic solvents exhibit antibacterial activity; it was ascertained that ethanol in which the product of our reaction was dissolved, did not contribute to the antibacterial activity (no zone of inhibition was obtained).The results ; and the formation of p-HCAM was confirmed using NMR and FTIR. Among the six bacterial strains used in this study, p-HCAM exhibited remarkable antibacterial activity against P. vulgaris and B. cereus.The PTAL catalyzed biotransformation of p-HCAM against species known to cause a number of diseases in humans including pneumonia, meningitis, bronchitis, food poisoning, and botulism. We also intend exploring the potential of using p-HCAM as a topical treatment agent or disinfectant and food additive in canned food material. This data would be important from fundamental and applied perspectives.Further work in this direction would involve a systematic study on antimicrobial activity of The laboratory work was primarily carried out by MM and Dr. PA. The manuscript was written by Dr. PA and Dr. GD. GD was the mentor. Dr. DB and Ms. JM provided their expertise for experimental work and analysis of results.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The prognosis of patients with advanced gastric cancer is poor. The goal of this study was to evaluate the efficacy and safety of combination therapy of cetuximab and S-1 combined with oxaliplatin (SOX) in Chinese patients with advanced gastric cancer.2 at the first infusion then 250 mg/m2 every week) was administered. For patients in both the control  and experimental groups, oxaliplatin (100 mg/m2) was administered intravenously on day 1, while S-1 (80 mg/m2/day) was given orally twice daily for 14 days. The endpoints of this study included progression-free survival, response rate, and disease-control rate.For patients in the experimental group (cetuximab in combination with SOX (Ce-SOX), 30 patients), once-weekly cetuximab (400 mg/mP = 0.225). The difference in disease-control rate was also statistically insignificant between the two groups . Median progression-free survival in the Ce-SOX group was significantly higher than that in the SOX group . The median overall survival of the Ce-SOX group and SOX group was 14.0 and 12.2 months, respectively (P = 0.043). The one-year survival rate for the Ce-SOX group was 57% compared to 40% in the SOX group. There was no statistical difference in the grade 3 or 4 adverse effects between the two groups.There was no statistically significant difference in response rate between the Ce-SOX and SOX groups (54.8% versus 44%, These findings suggest that the cetuximab combined with SOX regimen is feasible and shows promising efficacy with tolerable adverse effects in Chinese patients with advanced gastric cancer. Variouset al. .S-1 is an orally active derivative of 5-FU, which is a fourth generation oral fluoropyrimidine ,6, and tCetuximab is a recombinant, human/chimeric IgG1 monoclonal antibody (mAb) directed against the epidermal growth factor receptor (EGFR) . CetuximCetuximab has been evaluated in many phase II studies in patients with advanced GC either as monotherapy or combined with chemotherapy. In the first-line metastatic setting, several trials have evaluated combinations of cetuximab with different chemotherapy regimens -22 and aEpidermal growth factor receptor has been found to be overexpressed in gastric cancer ,25. Alth9, leukocyte count = 3 to 10 × 109, neutrophil count ≥1.5 × 109). Laboratory examination showed adequate liver function  and renal function . All patients provided written informed consent and this study was approved by Institutional Review Board of Henna Cancer hospital. All eligible patients were enrolled in this study between 1 August 2010 and 30 September 2012, and were randomly assigned into either the control group (26 patients) or the experimental group (30 patients).Fifty-six patients with histologically or cytologically proven unresectable gastric cancer or recurrence post-operation were enrolled. All of the patients had at least one measurable lesion by the Response Evaluation Criteria in Solid Tumors (RECIST) criteria, were ECOG performance status (PS) 0 to 2 and had anticipated life expectancies >3 months. Additionally, the patients were required to be chemotherapy naive or more than 6 months past the last adjuvant chemotherapy and to possess favorable bone marrow reservation  was administered. For patients in both the control and experimental groups, oxaliplatin (100 mg/m2) was administered intravenously on day 1. S-1 (80 mg/m2/day) was given orally twice daily for 14 days. All patients then took one week’s rest before the next cycle. Physical examination and blood analysis were performed at each cycle during chemotherapy. Responses of chemotherapy were assessed according to the RECIST criteria. Responses were classified as complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). Response rate was calculated as the sum of CR and PR. The disease-control rate was the sum of CR, PR, and SD. Six cycles of chemotherapy were given to all patients who showed response. Computed tomography and magnetic resonance imaging were used to assess tumor size. Second-line chemotherapy was given to patients who did not show response to the SOX or SOX + cetuximab (Ce-SOX) treatment. For grade 3/4 adverse events, the chemotherapy dose could be reduced if the symptoms were alleviated after management. Chemotherapy could be postponed if a patient’s condition still did not meet the criteria for chemotherapy treatment after adverse-event management.For patients in the experimental group, once-weekly cetuximab (400 mg/mPatients were followed up every 3 months until death or until the cutoff date of this study on 30 September 2012. The response rate (RR) was calculated as CR plus PR, time to progression (TTP) was defined as the period from the time of initial chemotherapy to PD and OS was defined as the period from the time of initial chemotherapy to death or the time of the final follow-up. Toxicity was assessed as grade 1 to 4 according to the US National Cancer Institute’s Terminology Criteria for Adverse Events V3.0.P <0.05 were regarded as statistically significant.Statistical analyses were conducted using SPSS 13.0 software. Progression-free survival (PFS) was calculated from the date of randomization to the time of disease progression, death, or September 30, 2012. PFS and OS were analyzed using the Kaplan-Meier method. The chi-squared test was used to compare the differences between groups. Results with Fifty-six patients were enrolled onto our study from August 2010 to September 2012. Patient characteristics are displayed in Table P = 0.071). The response rate in the Ce-SOX group was 54.8%, which was higher than the 44.0% in the SOX group, but failed to demonstrate statistical significance . The disease-control rate of the Ce-SOX group and SOX group was 87.1% and 76.0%, respectively    , b.i.d., with a schedule of 14 days on and 7 days off, while oxaliplatin was administered 130 mg/m2 on day 1.) that showed there were 2 (5.26%) CR, 12 (31.58%) PR, 15 (39.47%) SD, and 9 (23.68%) PD. The overall response rate and the disease control rate (DCR) was 36.84 and 76.31%, respectively. The TTP was 7.9 months. The median survival time (MST) was 10.2 months. These results demonstrated that the SOX regimen was effective and tolerable in the treatment of patients with AGC. The similar efficacies with less toxicity of SOX regimen in the other two studies indicate that the SOX regimen has the potential to replace current regimens [P = 0.007), 14.0 versus 12.2 months (P = 0.043), respectively). The one-year survival rate for the Ce-SOX group was 57% compared to 40% in the SOX group. These results suggest that cetuximab in combination with the SOX regimen shows significant therapeutic benefits to the Chinese patients with AGC. However, this study is limited by its small sample size. Future randomized, double-blind, large-scale clinical trials are warranted to clarify the role of this regimen in advanced gastric cancer.In 2013, Liu et al. conducteregimens . In the In conclusion, our study showed that cetuximab combined with the SOX regimen gave a favorable efficacy and safety profile in Chinese patients with advanced gastric cancer. Compared with the SOX regimen, this new regimen exerts significant therapeutic benefits to Chinese patients with advanced gastric cancer.AGC: advanced gastric cancer; Ce-SOX: cetuximab in combination with S-1 and oxaliplatin; CR: complete response; DCR: disease control rate; EGFR: epidermal growth factor receptor; GC: gastric cancers; GEJ: gastro-esophageal junction; mAb: monoclonal antibody; MST: median survival time; ORRs: objective response rates; OS: overall survival; PD: progressive disease; PFS: progression-free survival; PR: partial response; PS: performance status; RECIST: Response Evaluation Criteria in Solid Tumors; RR: response rate; SD: stable disease; SOX: S-1 and oxaliplatin; TTP: time to progression.The authors declare that they have no competing interests.All authors read and approved the final manuscript.


The factors that potentially affected the adsorption efficiency of the SA-PGA composite, including the initial concentration of REEs, the adsorbent dosage, and the pH of the solution, were investigated. 15 types of REEs in single and mixed aqueous solutions were used to explore the selective adsorption of REEs on gel particles. Scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) spectroscopy analyses of the SA and SA-PGA gel beads suggested that the carboxyl groups in the composite might play a key role in the adsorption process and the morphology of SA-PGA changed from the compact structure of SA to a porous structure after doping PGA. The kinetics and thermodynamics of the adsorption of REEs were well fit with the pseudo-second-order equation and the Langmuir adsorption isotherm model, respectively. It appears that SA-PGA is useful for recycling REEs from wastewater.With the exploitation of rare earth ore, more and more REEs came into groundwater. This was a waste of resources and could be harmful to the organisms. This study aimed to find an efficient adsorption material to mitigate the above issue. Through doping sodium alginate (SA) with poly-γ-glutamate (PGA), an immobilized gel particle material was produced. The composite exhibited excellent capacity for adsorbing rare earth elements (REEs). The amount of La Rare earth elements (REEs), also known as “Industrial Vitamins”, are widely used in numerous technological devices, such as superconductors, magnets, catalysts, and batteries. The amo2+,Ba2+), which makes the separation of SA more easy. One problem associated with particles is the low adsorption efficiency.Sodium alginate (SA) is a natural hydrophilic colloidal polysaccharide with an abundance of free carboxyl and hydroxyl groups distributed along its backbone. SA is generally isolated from brown seaweeds and bacteria. BecauseBacillus. PGA has unique physicochemical properties, such as film-forming ability, water-retention capacity, plasticity, and biodegradability:Qt=:Qt=Qt)AD) of REEs from the aqueous solution by the gel particles was calculated using Eq (Qt is the adsorption capacity of the gel particles for REEs (mg/g), Ci is the initial REE concentration (mg/L), Ct is the REE concentration in solution at time t (mg/L), and Ms is the dry weight of the gel particles (mg/L).Additionally, the adsorption rate (using Eq :AD=Ci−CQe and Qt (mg/g) are the sorption capacities at equilibrium and at time t, respectively, and k1 and k2 are the pseudo-first-order and pseudo-second-order sorption rate constants ((g/mg)/min), respectively.The kinetics for the adsorption of REE ions was analyzed using two kinetic models: pseudo-first-order and pseuThe Langmuir and Freundlich isotherm equations are generally used to describe the adsorption thermodynamics. After simplifying the equations, we obtain the following linear forms.Langmuir isotherm equation:Ce is the concentration of REE ions at equilibrium (mg/L), Qm is the maximum theoretical adsorption amount (mg/g), KL is the Langmuir adsorption equilibrium constant (L/mg), KF is the adsorption capacity of the particles (mg/g), and n is the affinity between the particles and adsorbate.Freundlich isotherm equation:3+ and Ce3+ exhibited similar behavior on these two gels. As the ion concentration increased, the adsorption rate decreased after a plateau period. In contrast, the adsorption amount increased in an approximately linear manner before the excess of adsorption sites. Moreover, note that the saturated adsorption amount of SA (57.82 mg/g) was only 35.47% of that of SA-PGA (163.02 mg/g). This result indicated that doping with PGA could greatly improve the efficiency for adsorbing REEs ions.The results regarding the effect of initial concentration of REE ions are presented in 3+ removal efficiency was 17.62%, whereas at the highest SA particle dosage, the adsorption rate reached 39.88%. In contrast, these two data of SA-PGA were 44.05% and 99.70%, respectively. Thus, this result indicated that SA-PGA particles are a more effective type of adsorbent.The effect of adsorbent dosage on the adsorption of REEs is shown in + will compete with rare earth cations for binding sites. Therefore, it is difficult for REEs to approach the adsorption sites. On the contrary, under basic pH conditions, more sites with negative charges will be exposed toward the adsorbate. Thus, REE ions with positive charges can be adequately attracted. However, under alkaline pH conditions, the REE ions will form hydroxide precipitates. The initial pH was adjusted to pH 2.0–7.0. + had the dominant superiority compared with other cations. The interaction was weak between adsorbent and R3+. The concentration of Ca2+ reached about 64 mg/L. In pH4.0–5.0, main cations were H+, Ca2+ and R3+. Accompanied by the loss of dominance, Ca2+ released and R3+ were adsorbed.The pH may be the most important parameter in the adsorption process because it can affect not only the surface charge of the adsorbent but also the degree of ionization and the speciation of the adsorbate during the adsorption process. NormallUnder natural conditions, REEs exist in a mixed state. Therefore, it is necessary to investigate the selectivity of REEs on SA-PGA gel particles. To determine the optimal adsorption conditions, response surface experiments based on single-factor tests were conducted under the following conditions: initial concentration of REEs, 220 mg/L; adsorbent dosage, 0.0562 g; pH 5.0; 150 rpm; and 30°C. In the single solution of 15 rare earth elements , the ads-1ν(C-OH) and 1626cm-1νas(COO-), which are assigned to stretching vibrations of the carboxyl group[-1 δ(-OH)+ν(COO-)asym and 1419cm-1 ν(COO-)sym in SA-PGA. Under normal circumstance, absorption peak of carbonyl (in COOH) should appear at 1700–1725cm-1. From the view of coordination chemistry, the type of carboxyl in SA-PGA gel was a single-tooth ligand with Ca2+. -1 attributed to ν(CN). This suggested that PGA was successfully immobilized in the SA gel. The other peaks of the SA-PGA gel exhibited no difference with respect to those of the SA gel, which indicated that PGA was very likely fixed through a mode of non-chemical interaction, such as electrostatic attraction or hydrogen bonding[3+, the peak at 1615 cm-1 attributed to δ(-OH)+ν(COO-)asym had shifted to a stronger wavenumber 1629cm-1. It showed that carboxyl in SA and PGA participated in the coordination with REEs ions. Moreover, 1733cm-1 assigned to ν(C = O in COOH) absorption peak only appeared in SA-PGA-La-HCl spectrum. It showed that free-COOH transformed into-COOCa in the process of preparing SA-PGA gel. This result suggested that the carboxyl group coordinated with the rare earth ions both in SA and PGA. The adsorption mechanism was likely to be cation exchange between-COOCa and R3+.The FT-IR spectroscopy  results are shown in xyl group. There an bonding. After tDistinct differences were observed in the morphologies between SA and SA-PGA gel beads . Fig 4A r2) of the models were determined by linear regression analysis and are listed in t/Qt vs. t  were calculated by plotting Ce/Qe vs. Ce and ln Qe vs. ln Ce and are presented in KL values are the key parameters for calculating the thermodynamics constants. Additionally, it is generally stated that values of n ranged from 2to 10 representing good adsorption. The results indicated that the values of n were all greater than 2, which implied that REE ions were favorably adsorbed by the gel. By comparing the adsorption equilibrium constants and the adsorption affinities of these two types of materials, it was observed that the combination with poly-γ-glutamate was beneficial in enhancing the adsorption efficiency. Through the combination of internal structure and composition, the thermodynamic features of SA-PGA exhibited superior mass transfer ability.From the experimental data, the adsorption equations were obtained using Eqs  and 6)..6). The ΔG), enthalpy (ΔH), and entropy (ΔS) [ΔH and ΔS were calculated from the slope and intercept of the linear plot of ln (Qe/Ce) vs. 1/T according to Eq  . The effng to Eq , i.e., tusing Eq .lnQeCe=ΔG were negative, the process could occur spontaneously at room temperature. The absolute values of ΔG increased with increasing temperature. Additionally, these values showed that the increasing temperature improved the feasibility of the adsorption process[ΔH were positive, the adsorption was an endothermic process. In other words, the higher the temperature of the system, the better the result. Viewed from values of ΔH (20.1~418.4 kJ/mol), the process was more inclined to chemical adsorption[ΔS>0.The thermodynamic constants are shown in n process. Moreovedsorption. The deg2+ during the adsorption-desorption process[3+. Additionally, a new bright silver metallic luster appeared on the surface of the adsorbents.For the enrichment or circulation of REEs, desorption is another key issue. In this work, 0.05 M hydrochloric acid solution was used as the eluent. Additionally, the desorption rate was greater than 99.00%. During the first ten adsorption-desorption cycles, these two adsorbents could retain their adsorption capacities . Howevern process, the cor3+ according to FTIR. The SA-PGA particle exhibited a preferential effect on both high and low concentrations of REEs. It still maintained good adsorption capability after 10 adsorption-desorption cycles. SA-PGA can be potentially used in recycling of REEs.This study showed that SA-PGA gel particles possessed a considerable adsorption capacity for REEs(Ⅲ). High or low pH conditions inhibited adsorption. From Sc to Lu, adsorbent showed good adsorption on them. The pseudo-second-order kinetic model agreed well with the dynamic adsorption. The thermodynamic parameters indicated that the adsorption was an endothermic process and could occur spontaneously. The adsorption mechanism might be cation exchange between—COOH and RS1 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.